Yang et al. BMC Plant Biology 2012, 12:140http://www.biomedcentral.com/1471-2229/12/140

RESEARCH ARTICLE Open Access

Identification of the dehydrin gene family fromgrapevine species and analysis of theirresponsiveness to various forms of abiotic andbiotic stressYazhou Yang1,2,3, Mingyang He1,2,3, Ziguo Zhu1,2,3, Shuxiu Li1,2,3, Yan Xu1,2,3, Chaohong Zhang1,2,3,Stacy D Singer4 and Yuejin Wang1,2,3*

Abstract

Background: Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and alsoparticipate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHNgene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderatelyresistant to powdery mildew.

Results: Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequenceidentity between the two species but little homology between the genes themselves. These genes weredesignated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predictedto be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number offunctional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat,embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). Itwas expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a secondround of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V.vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited noresponsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetativetissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development atextremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particularcis-elements within the promoter regions of each gene was positively correlated with their expression profiles.

Conclusions: The grapevine DHN family comprises four divergent members. While it is likely that their functionsoverlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our resultssuggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elementsin the promoter regions of the DHN genes.

Keywords: Grapevine, Dehydrin, Stress-induced expression, Powdery mildew, Promoter

* Correspondence: wangyj@nwsuaf.edu.cn1College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100,China2Key Laboratory of Biology and Genetic Improvement of Horticultural Crops(Northwest Region), Ministry of Agriculture, Northwest A&F University,Yangling, Shaanxi 712100, ChinaFull list of author information is available at the end of the article

© 2012 Yang et al.; licensee BioMed Central LtCommons Attribution License (http://creativecreproduction in any medium, provided the or

d. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

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BackgroundDehydrins (DHNs) are a class of hydrophilic, thermostablestress proteins with a high number of charged amino acidsthat belong to the Group II Late Embryogenesis Abundant(LEA) family. Genes that encode these proteins areexpressed during late embryogenesis, as well as in vegeta-tive tissues subjected to drought, low temperature andhigh salt conditions [1-3]. Intriguingly, over-expression ofDHN genes in transgenic plants has been found to en-hance resistance of the transgenic lines to various adverseenvironments, such as cold, drought, salinity and osmoticstress [4-7], which has raised significant interest in theirputative application for crop improvement. Furthermore,it has recently been shown that reduced levels of dehy-drins in transgenic Arabidopsis seeds leads to reducedseed longevity [8], emphasizing their importance to seedsurvival in addition to their influence on vegetative stresstolerance.While it is generally accepted that DHNs function to

protect cells from damage caused by stress-induced dehy-dration [9], their precise mechanism remains elusive.However, it has been proposed that they may carry outtheir function through membrane stabilization by actingas chaperones to prevent the aggregation and/or inactiva-tion of proteins under dehydration or high temperatureconditions [5,10,11].Classification of DHNs is based upon structural fea-

tures of the proteins, such as the presence and copynumber of certain conserved motifs, such as the K-, S-,and Y-segments. To date, these proteins have beendivided into 5 subclasses, including YnSKn, YnKn, SKn, Kn

and KnS [12]. All DHNs possess at least one K-segment(EKKGIMDKIKEKLPG), which is generally located at theC-terminal end of the protein and has the ability to forman amphipathic helix-like structure that may play a rolein its interaction with membranes and proteins [13,14].The S-segment consists of a track of serines that can bemodified through phosphorylation and may function inthe regulation of protein conformation and ion-bindingactivity [15-17]. The Y-segment (DEYGNP) is locatednear the N-terminus and shows partial amino acid iden-tity to the nucleotide binding site motif of chaperoneproteins from various organisms [12].Several other conserved regions have also been identi-

fied in a subset of DHNs. For example, lysine-rich seg-ments (Lys-segments) contain a cluster of lysines thatare generally located between the S- and K-segments[18,19] and have been suggested to participate in thebinding of DHNs to DNA or RNA [19]. Nuclearlocalization signals (NLSs), which bear an RRKK motif,have been found specifically in YSKn-type DHNs andplay a role in their localization to the nucleus [15,16,20].Furthermore, phosphorylation has been found to be animportant factor for substrate binding of DHNs

[16,17,21], and recently a His switch has been found tobe involved in the regulation of membrane binding ofthe Arabidopsis thaliana DHN, LTI30 [22].At the functional level, DHN family members often

exhibit sub-functionalization, with different genes dis-playing differential expression profiling throughout de-velopment and under stress conditions. For example,while both LTI29 (SK2) and LTI30 (K6) were up-regulated in Arabidopsis under low temperature con-ditions, only LTI30 was up-regulated following salttreatments [1]. Similarly, while ten barley DHNs werefound to be up-regulated by drought, only three wereup-regulated by low temperatures [23], and in Oleaeeuropaea, although expression levels of 40 kDa and42 kDa DHNs increased in response to various stressors(including dehydration, high salinity, and wounding),16 kDa and 18 kDa DHNs were mainly induced by saltstress [24]. These differences in expression patterns im-ply functional diversification within this gene family;however, at present, the relationship between subgroupclassification and expression profile is unclear [9].Grapevine is one of the most important fruit crops in

the world and while the majority of grape varieties aredirectly cultivated from Vitis vinifera L., this species isrelatively susceptible to powdery mildew (Erysiphe neca-tor). Conversely,V. yeshanensis is a wild species of grapenative to the Yanshan mountain in Hebei province,China, that is highly tolerant to both cold and drought[25,26], and is also resistant to E. necator [27]. Previ-ously, two highly similar putative Y2SK2-type DHN genes(DHN1a and DHN1b) were identified in V. vinifera andtheir expression was found to be induced by multipletypes of stress, such as drought, cold and high salinity[28,29]. In this study, we aimed to identify the membersof DHN gene family in V. vinifera, as well as their hom-ologous equivalents in V. yeshanensis. In doing so, wewere able to investigate the functional divergence of thisgene family in these two species through comparisons oftheir expression profiles and putative physicochemicalcharacteristics. Furthermore, we also assessed possiblerelationships between specific cis-elements within DHNpromoter sequences and the regulation of their expres-sion under various conditions.

ResultsIdentification of DHN family members in V. vinifera and V.yeshanensisA 280-bp fragment of a DHN cDNA was cloned fromdrought-treated leaves of V. yeshanensis acc. Yanshan-1using differential display reverse transcription-PCR(DDRT-PCR; Additional file 1). Subsequently, the full-length sequence was determined using 5’ rapid amplifi-cation of cDNA ends (RACE) and was termed VyDHN1[GenBank:JF900497]. The putative protein sequence of

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this gene was then utilized to detect DHN genes fromthe published V. vinifera cv. Pinot Noir clone PN40024genome sequence [30] via BLAST analysis. Four DHNgenes were identified, all of which contained a K-seg-ment. These genes were designated VvDHN1 (corre-sponding in sequence to the previously identified V.vinifera DHN1a) [GenBank:XM_03631828], VvDHN2[GenBank:XM_002285883], VvDHN3 [GenBank:CAN73166], and VvDHN4 [GenBank:XM_002283569].The three remaining DHN genes were cloned from V.

yeshanensis acc. Yanshan-1 seed-specific cDNA usingprimers derived from the V. vinifera sequences and weredesignated VyDHN2 [GenBank:JQ408442], VyDHN3[GenBank:JQ408443], and VyDHN4 [GenBank:JQ408444]. While only 25 amplification cycles wererequired to clone both VyDHN2 and VyDHN4, 40 cycleswere required in the case of VyDHN3. Subsequently, allfour V. yeshanensis genes were amplified from genomicDNA to identify intronic regions [GenBank:JF896520,JF896556, JF896557, and JF896558, respectively]. In bothspecies, all four DHN genes consisted of two exons sepa-rated by one intron present within the S-segment.In terms of nucleotide similarities, virtually no se-

quence identity was detected between the four DHNcoding sequences. However, high levels of homologywere noted between matching genes belonging to thetwo different species, with 97% (DHN4) and 99%(DHN1, DHN2 and DHN3, respectively) identity at thenucleotide level. In terms of chromosomal localization,while VvDHN1 and VvDHN2 were located on chromo-somes 4 and 18, respectively, VvDHN3 and VvDHN4were mapped to chromosome 3 in opposite orientations(Figure 1).

Characterization and comparison of deduced DHNproteinsProtein sequences were deduced from the correspondingV. vinifera and V. yeshanensis DHN cDNA sequences,and were composed of 130–206 amino acids exhibiting97-99% identity at the amino acid level between the twospecies. Both K- and S-segments were found to be highlyconserved between members of the V. vinifera and V.yeshanensis DHN families, whereas remaining regionsdisplayed relatively low amino acid identity between thefour genes. Furthermore, while NLS domains were iden-tified in both DHN1 and DHN4 proteins, a Lys-rich seg-ment was only present in DHN2 (Figure 2). Based onthe presence and number of K-, S- and Y-motifs (Fig-ure 2), the four DHNs from each species were classifiedas either Y2SK2- (DHN1), SK2- (DHN2), SK3- (DHN3),or Y3SK2-type (DHN4) proteins (Figure 2; Table 1).All members of the DHN family in the two grapevine

species analyzed were found to be highly hydrophilic,with GRAVY values ranging from −0.959 to −1.527 and

theoretical pIs from 5.20 to 9.36 (Table 1). DHN1 andDHN4, which were YnSKn-type DHNs, possessed ahigher pI than the SKn-type DHNs (DHN2 and DHN3)in both species. In terms of acidity, our analyses indi-cated that DHN1 was the sole basic protein, whileDHN2 was the most acidic.Many phosphorylation sites were also predicted within

each of the DHN protein sequences analyzed, withDHN1 and DHN4 containing a higher number of puta-tive protein kinase C (PKC) phosphorylation sites thancasein kinase 2 (CK2) phosphorylation sites, and DHN2and DHN3 containing a higher number of CK2 sitesthan PKC sites (Table 1; Figure 2). In addition, a recentlyidentified conserved motif (LXRXXS) phosphorylated byan Snf1-related kinase (SnRK2-10) [31] was identified inboth DHN1 and DHN2 proteins.While all four grapevine DHNs were found to be rich

in disordered regions and contained relatively few helixor strand motifs (see Additional file 2), the YnSKn-typeproteins (DHN1 and DHN4) displayed the highest dis-order index and least helix/strand-motifs. Additionally,of the few helix motifs identified, most were locatedwithin K-segments, which is consistent with the findingsof a previous study of DHN protein structure [14].

Phylogenetic analysis of V. vinifera and V. yeshanensisDHNsTo date, DHN families from both barley and Arabidopsishave been thoroughly characterized at the genomic level[18,32-35]. Therefore, to provide a further understandingof the relationships between the V. vinifera and V. yesha-nensis DHNs, we conducted a phylogenetic analysis ofthese genes via comparison with those from barley andArabidopsis. Overall, we found the number of DHN genesin the grapevine species (four) to be smaller than that ineither barley (thirteen) or Arabidopsis (ten). Based on ourphylogenetic results, the DHNs could be divided into fourgroups, corresponding to YnSKn-, SKn-, Kn-, and KS-typeproteins (Figure 3), where the Arabidopsis YK-type DHN(At4g39130) was included within the YnSKn group. Asexpected from our classification of the grapevine DHNsequences based on the presence of various conservedsegments, the grapevine DHN1 and DHN4 proteins weregrouped together with the YnSKn-type DHNs of Arabidop-sis and barley, while the grapevine DHN2 and DHN3 pro-teins were grouped with the SKn-type DHNs ofArabidopsis and barley. Interestingly, both grapevine spe-cies lacked KS- and Kn-type DHNs; groups which arepresent in both barley and Arabidopsis.

Expression profiles of DHN transcripts in various tissuesand developmental stagesTo elucidate the physiological functions of differentmembers of the DHN family in V. vinifera and V.

Figure 1 Predicted structure and chromosomal localization of V. vinifera DHN genes. Chromosomal localization of the four DHN genes wasdetermined from the V. vinifera cv. Pinot Noir clone PN40024 genome sequence. Exons are represented as gray boxes, with respective lengthsindicated within each box. Lengths of introns, which are denoted by a line between exons, are indicated above each line. Arrows indicatedirection of transcription in each case.

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yeshanensis, the expression of DHN genes was investi-gated at veraison in roots, stems, leaves, seeds, and fruitpeels using semi-quantitative RT-PCR (Figure 4). Bothspecies exhibited highly similar expression profiles fortheir matching DHN genes. Results suggested that undernormal growth conditions, DHN1 was mainly expressedin seeds, with very low levels present in the roots. DHN2was constitutively expressed in all tissues; however,weaker levels of expression were noted in leaves andstems than in the other organs tested. DHN3 was un-detectable under the current experimental conditions,suggesting that it was not expressed, or was at levels toolow to be detected using this method, in these tissues.The DHN4 genes, on the other hand, were expressedspecifically in seeds. These results indicate that the fourDHN genes that make up the V. vinifera and V. yesha-nensis DHN gene families, respectively, exhibited verydistinct expression patterns in the organs tested.Intriguingly, reactions in which DHN1, DHN2 and

DHN4 were amplified often exhibited two bands: onethat corresponded in size to a spliced transcript, andone that seemed to be closer in size to the band amp-lified from genomic DNA (ie. containing an unsplicedintron). The fact that no amplification products wereobtained in negative control reactions lacking reversetranscriptase confirmed that template RNA was free from

contaminating genomic DNA. Therefore, it seems thatthese larger transcript variants resulted from the pres-ence of unspliced DHN transcript variants (either mRNAor pre-mRNA) within the total RNA pool.To gain a more precise understanding of grapevine

DHN expression during the process of seed develop-ment, inflorescences and berries of V. vinifera were har-vested from flowering to veraison and used for qRT-PCRexpression analyses. DHN1 and DHN2 were found to beexpressed in floral buds even 6 d before opening of theflowers (Figure 5A and B), after which time their tran-scripts decreased during the middle stages of embryo-genesis, and were strongly up-regulated once againduring later stages of embryogenesis. Low levels ofDHN3 were detected in both mid- and late-stages ofseed development (Figure 5C) while DHN4 transcriptswere only detectable during late stages of embryogen-esis, with peak expression developing just prior to verai-son (Figure 5D).

Response of DHN gene expression to various abiotic andbiotic stressesIn an attempt to determine whether DHN1, DHN2,DHN3 and DHN4 exhibited stress-responsiveness, weanalyzed the expression levels of all four genes in theleaves of three V. vinifera and V. yeshanensis plants,

Figure 2 Sequence alignment of DHN proteins from V. yeshanensis and V. vinifera. Y-segments, S-segments and K-segments are denotedby gray shading. NLS and Lys-rich segments are framed by a black line. Phosphorylation sites are in bold, with PKC sites underlined with a singleline, CK2 sites in italics, and SnRK-10 sites underlined with a dotted line.

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respectively, that had been subjected to various stressconditions using real-time qRT-PCR. Neither DHN3 norDHN4 from either species exhibited detectable levels of

Table 1 Characteristics of DHN proteins in V. yeshanensis and

Name Type No. of Residues MW (kDa) pI GRAVY

VyDHN1 Y2SK2 130 13.9 9.36 −1.425

VvDHN1 Y2SK2 130 13.9 9.27 −1.459

VyDHN2 SK2 205 23.4 5.21 −1.527

VvDHN2 SK2 206 23.5 5.20 −1.514

VyDHN3 SK3 166 18.8 5.81 −1.736

VvDHN3 SK3 166 18.9 5.92 −1.739

VyDHN4 Y3SK2 191 20.1 6.35 −0.959

VvDHN4 Y3SK2 191 20.1 6.26 −1.030

MW (molecular weight), pI (isoelectric point) and GRAVY (grand average of hydroparefer to specific phosphorylation sites. Expression information is based on the RT-PC

expression under any of the conditions tested here, in-cluding drought, cold, heat, or E. necator infection; there-fore, only DHN1 and DHN2 will be discussed further in

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PKC No CK2 No SnRK2 No Expression

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thy) were predicted based on amino acid composition. PKC, CK2 and SnRK2R experiments described in Figures 4 through 7.

Figure 3 Phylogenetic relationships between grapevine, barley and Arabidopsis DHN proteins. The unrooted dendrogram wasconstructed with the PhyML tool using the maximum likelihood method based on a complete protein sequence alignment of DHNs fromArabidopsis thaliana (At), Hordeum vulgare (Hv), V. vinifera (Vv), and V. yeshanensis (Vy). The bootstrap value is given for each node. GenBankaccession numbers are as follows: VvDHN1 [XM_03631828], VvDHN2 [XM_002285883], VvDHN3 [CAN73166], VvDHN4 [XM_002283569], VyDHN1[JF900497], VyDHN2 [JQ408442], VyDHN3 [JQ408443], VyDHN4 [JQ408444], At1g20440 [AY114699], At1g20450 [AF360351], At1g54410[NM_104319], At1g76180 [AF339722], At2g21490 [BT000900], At3g50970 [NM_114957], At3g50980 [NM_114958], At4g38410 [NM_120003],At4g39130 [NM_120073], At5g66400 [AY093779], HvDhn1 [AF043087], HvDhn2 [AF181452], HvDhn3 [AF181453], HvDhn4 [AF181454], HvDhn5[AF181455], HvDhn6 [AF181456], HvDhn7 [AF181457], HvDhn8 [AF181458], HvDhn9 [AF181459], HvDhn10 [AF181460], HvDhn11 [AF043086],HvDhn12 [AF155129], HvDhn13 [AY681974].

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this section. In the case of drought conditions, althoughwe found that DHN1 was induced by this treatment inboth species (Figure 6A), we noted slight differences be-tween the two species. While DHN1 transcripts increasedin V. yeshanensis between 1–2 d after the drought treat-ment began and reached a maximum induction of a 237-fold increase compared to baseline expression levels 5 dafter treatment, its homologue in V. vinifera was delayedin its exhibition of a response (between 2–3 d) andreached a higher maximum induction of 366-fold com-pared to baseline levels. Conversely, DHN2 did not appear

to respond to drought treatment in either of the two spe-cies tested (Figure 6B). Interestingly, transcripts of bothDHN1 and DHN2 also exhibited up-regulation in bothgrapevine species shortly after rehydration (Figure 6 Aand B), with a maximum level of expression achieved afterapproximately 2 h.Following cold and heat treatment, both DHN1 and

DHN2 were induced in V. vinifera and V. yeshanensis.While DHN2 transcripts increased gradually between0 h and 48 h following initiation of cold stress in bothspecies, DHN1 transcripts exhibited a more sudden

Figure 4 Expression of DHNs in various organs of V. yeshanensis and V. vinifera. Total RNA was isolated from root (R), stem (St), leaf (L),seed (Sd) and fruit peel (P) at veraison, and was quantified using a Nanodrop spectrophotometer (Nanodrop Products, Wilmington, DE, USA).1 μg DNase-treated total RNA was used as template for first-strand cDNA synthesis in a final volume of 20 μl, and subsequently 1 μl of thisreaction was utilized for PCR amplification in a volume of 25 μl. Genomic DNA (D) was utilized as the positive control. RNA without reversetranscriptase was used as the negative control. The grapevine actin1 fragment was amplified as an internal control. 15 μl of PCR products wereseparated on a 1.5 % agarose gel containing ethidium bromide in each case.

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onset of up-regulation in response to cold beginning be-tween 6–12 h after treatment (Figure 6 C and D). In thecase of heat stress, both DHN1 and DHN2 transcriptsincreased to maximum levels 24 hours following the ini-tiation of treatment in both species, and then subse-quently decreased (Figure 6 E and F).To determine whether grapevine DHNs were respon-

sive to biotic stress, the levels of DHN gene expressionwere tested in the leaves of V. vinifera and V. yeshanen-sis inoculated with E. necator, which is the causativeagent of grapevine powdery mildew. Results suggestedthat only DHN1 was induced by E. necator, wherebytranscripts increased gradually to a maximum at 3 dpost-inoculation (dpi) and then decreased slowly afterthis point in both species. Interestingly, V. yeshanensisexhibited a higher peak level of expression (18-fold in-crease compared to expression levels immediately priorto treatment) than V. vinifera (10-fold increase), as wellas an additional sharp increase in DHN1 expression 6dpi (Figure 6G). In contrast, DHN2 did not appear to re-spond to E. necator inoculation in either grapevine spe-cies (Figure 6H).

Response of DHN gene expression to levels of varioussignaling moleculesThe responses of plants to abiotic and biotic stress aregenerally mediated by abscisic acid (ABA) and salicylicacid (SA)/jasmonic acid (JA), respectively. To determinewhether the induction of grapevine DHNs under stressconditions was related to any of these signaling mole-cules, DHN expression was investigated in leaves fromthree V. vinifera plants treated with ABA, SA or MeJA,respectively. As was the case for studies involving abioticand biotic stress treatment, no detectable levels ofDHN3 or DHN4 expression could be detected in leavestreated with any of these chemicals. While DHN1 wasinduced by ABA, SA and MeJA (Figure 7A), the most

considerable up-regulation (160-fold compared to base-line levels) was noted 8 h following application of ABA.DHN1 transcripts from leaves treated with MeJAreached a maximum induction of ~20-fold compared tobaseline levels approximately 4 h following application,whereas a similar level of induction was reached 8 h fol-lowing treatment with SA. In the case of DHN2 tran-scripts, expression reached a maximum 5-fold inductioncompared to baseline levels 4 h after application ofABA. Although slight modifications were also noted inDHN2 expression levels following SA and MeJA applica-tions, respectively, they differed only slightly fromchanges observed in untreated samples (Figure 7B).

Comparison of cis-regulatory elements in the upstreamregulatory regions of grapevine DHN genesAll four DHN promoters (including 1500-bp of sequenceupstream of the translational start codon) were clonedfrom V. yeshanensis acc. Yanshan-1 [GenBank: JF899925for VyDHN1, GenBank: JX110839 for VyDHN2, Gen-Bank: JX110840 for VyDHN3, and GenBank: JX110841for VyDHN4] using primers derived from the corre-sponding V. vinifera sequences. High levels of homologywere detected between matching promoters from thetwo different species, with 94% (DHN1), 96% (DHN2),and 92% (DHN4) identity at the nucleotide level. Thepromoter of DHN3, on the other hand, only exhibited84% identity between the two species due to a 205-bpdeletion in this sequence from V. yeshanensis.To elucidate whether the differential expression pat-

terns of the four grapevine DHN genes correlate withtranscriptional regulation via their promoters, upstreamregions of each gene from V. yeshanensis and V. viniferawere scanned for putative cis-regulatory elements usingthe PlantCARE database [36]. Nucleotide sequences in-cluding 1500-bp upstream of each start codon wereobtained from the V. vinifera cv. Pinot Noir clone

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Figure 5 Expression of DHNs in V. vinifera during seed development. Total RNA was isolated from floral buds harvested 6 days prior toflower opening (−6 daf), flowers were harvested on the day of flower opening (0 daf), young berries were harvested 6 daf, and seeds wereharvested from 12–66 daf. Transcript levels of DHN1, DHN2, DHN3, DHN4 normalized to the levels of the internal control, actin1, were determinedusing real-time qRT-PCR analysis. Each block represents the mean relative fold-change compared to baseline levels of expression from threeexperiments, while bars indicate standard deviations. In the case of DHN1, DHN2 and DHN4, baseline expression levels were those measured at−6 daf. In the case of DHN3, baseline expression was set to that measured at 18 daf as this was the first time point at which expression wasnoted. Times indicating flowering and veraison are indicated by arrows.

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PN40024 genome database [30] , while those from V.yeshanensis were obtained directly via cloning, and cis-regulatory elements were classified into three groupsaccording to their potential responsive functions: abioticstress-related elements, biotic stress-related elements,and seed development-related elements. Abiotic stress-related elements comprised ABA-responsive elements(ABRE), dehydration-responsive elements (DRE), heatshock-responsive elements (HSE), and low temperature-responsive elements (LTR). Biotic stress-related elementsincluded MeJA-responsive elements (MeJA-RE), salicylicacid-responsive elements (TCA), as well as stress- and

defense-responsive elements (TC-rich repeats). Seeddevelopment-relatedelementscomprisedonlyendosperm-specific expression elements (Skn-1 motif ).All cis-regulatory elements found in the V. yeshanensis

and the V. vinifera DHN promoters, which exhibited asimilar composition and distribution of putative regula-tory elements between corresponding promoters, areshown in Table 2. There were obvious differences in theabundance and distribution of cis-regulatory elements inthe four DHN promoters analyzed (Figure 8). TheVyDHN1 and VvDHN1 promoters had the most diversecollection of putative cis-regulatory elements, including

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Figure 6 (See legend on next page.)

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(See figure on previous page.)Figure 6 Expression profiles of grapevine DHN1 and DHN2 under abiotic and biotic stress. Total RNA was extracted from the leaves of V.yeshanensis and V. vinifera treated with drought-rehydration (A, B), 4 °C (C, D), 40 °C (E, F), and inoculation with E. necator (G, H). Samples weretaken at the indicated times, with time zero samples harvested immediately prior to treatment. Normalized transcript levels of DHN1 and DHN2were determined by real-time qRT-PCR analysis, with the actin1 gene serving as an internal control. Each point represents the mean relative fold-change compared to baseline levels of expression from three experiments, while bars indicate standard deviations. Baseline expression levelswere those measured just prior to treatment at time= 0. Asterisks (*) and number signs (#) indicate significant increases (p < 0.05) in expressionlevels of target transcripts from V. yeshanensis and V. vinifera, respectively, compared to the mock-treated controls.

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several involved in stress-response, seed development,and hormone signaling (Table 2). In the case of theVyDHN2 and VvDHN2 promoters, HSE, LTR and TCAelements were lacking when compared to VyDHN1 andVvDHN1. Only a small number of potential cis-elementswere identified in the VyDHN3, VvDHN3, VyDHN4 andVvDHN4 promoters. In the VvDHN3 promoter, anABRE, Skn-1 and two TC-rich elements were scatteredevenly throughout the promoter, while the ABRE elem-ent was absent in that of VyDHN3. In VvDHN4, twoABREs, a Skn-1 and a MeJA-RE were concentrated inthe 3’ region of the promoter, while a TCA element waspresent in the 5’ region. A similar distribution was notedin the VyDHN4 promoter, with an additional Skn-1motif present 765-bp upstream of the translational startcodon.

DiscussionDehydrins are believed to play a fundamental role in theresponse of plants to various abiotic and biotic stresses.They make up a multigene family with 10 members inArabidopsis [18,35], 8 members in rice [45], 13 membersin barley [23], and 11 members in poplar [46]. However,only 4 DHN genes were identified in the published V.vinifera genome sequence [30,47], including two YnSKn-type DHNs (DHN1 and DHN4) and two SKn-type DHNs

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Figure 7 Expression profiles of grapevine DHN1 and DHN2 followingleaves of V. vinifera sprayed with 50 μM MeJA, 100 μM SA and 100 μM ABAsamples harvested immediately prior to spraying. Normalized transcript levwith the actin1 gene serving as an internal control. Each point represents texpression from three experiments, while bars indicate standard deviationstreatment at time= 0. Asterisks (*), number signs (#) and reference marks (※target transcripts from V. vinifera treated with ABA, SA and MeJA, respective

(DHN2 and DHN3). Neither Kn- nor KS-type DHNswere found in this species, which differs from the DHNgene families from other plant species characterized todate and suggests that these types of genes may havebeen lost in grape species (Figure 2).

Expansion of the DHN family has generally occurredthrough tandem duplication events and whole-genomeduplications. For example, At1g20440/At1g20450,At3g50970/At3g50980, and At4g38140/at4g39130 arosefrom tandem duplications, while At1g20450/At1g76180,At2g21490/At4g39130, and At3g50970/At5g66400 arosefrom a whole-genome duplication event in Arabidopsis[18], which together resulted in an increase of 6 DHNgenes. Similarly, whole-genome and tandem duplicationevents were responsible for an increase of 3 and 2 DHNgenes, respectively, in poplar [46]. At least 3 DHN genesarose from tandem duplication events in rice [45], and itis possible that the two clusters of DHN genes on chro-mosomes 5 H and 6 H in H. vulgare, respectively [32],which show a high level of sequence identity within eachcluster, may have resulted from tandem duplicationevents.While the genomes of poplar, rice and Arabidopsis

have undergone at least one recent whole-genome dupli-cation event, the grapevine genome has not [30]. In-stead, the four grapevine DHNs have likely arisen from

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treatment with various hormones. Total RNA was extracted from the(A, B). Samples were taken at the indicated times, with time zero

els of DHN1 and DHN2 were determined by real-time qRT-PCR analysis,he mean relative fold-change compared to baseline levels of. Baseline expression levels were those measured just prior to) indicate significant differences (p < 0.05) in expression levels ofly, compared to the mock-treated controls.

Table 2 Regulatory elements involved in stress-, pathogen- and embryogenesis-responsiveness in grapevine DHNpromoter regions

Cis-element Sequence Number of cis-elements* Function References

DHN1 DHN2 DHN3 DHN4

ABRE CACGTG 4/4 6/6 0/1 2/2 Abscisic acid responsiveness [37]

DRE ACCGAC 1/1 1/1 0/0 0/0 Drought and cold responsiveness [38]

HSE AGAAAATTCG 1/2 0/0 0/0 0/0 Heat stress responsiveness [39]

LTR CCGAAA 1/1 0/0 0/0 0/0 Low-temperature responsiveness [40]

TC-rich repeats ATTTTCTTCA 0/0 0/0 2/2 0/0 Stress and defense responsiveness [41]

MeJA-RE CGTCA 1/1 2/3 0/0 1/1 MeJA-responsiveness [42]

TCA element CCATCTTTTT 1/1 1/0 0/0 1/1 Salicylic acid responsiveness [43]

Skn-1_motif GTCAT 2/2 1/1 1/1 2/1 Endosperm expression [44]* The first number indicates the number of cis-regulatory elements within the V. yeshanensis DHN promoter while the second number indicates the number ofcis-regulatory elements within the V. vinifera DHN promoter.

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an ancestral genome [30], which is consistent with thelow level of sequence similarity between the foursequences. However, DHN3 and DHN4 lay in close prox-imity on chromosome 3 in V. vinifera, which impliesthat one of them may have arisen through a tandem du-plication event despite their low level of identity. There-fore, it seems that the relatively low number of DHNgenes in grapevine may simply be due to a lack of dupli-cation events in this genus. Indeed, it has been suggestedthat gene family expansion in grapevine has been select-ive, occurring mainly in those genes involved in aromaticfeatures [30].In silico characterization of V. vinifera and V. yesha-

nensis DHN protein sequences suggested they were allhighly hydrophilic and disordered, but with distinct dif-ferences in pI, kinase specificity and content of func-tional motifs. The two YnSKn-type DHNs (DHN1 andDHN4) possessed a higher pI than the SKn-type DHNs(DHN2 and DHN3). Since positively charged DHN pro-teins bind negatively charged membranes with a high af-finity [13], it follows that the YnSKn-type DHNs, andespecially DHN1, could very well bind with the cellmembrane in grapevine. It has been suggested that thebinding of DHNs to membranes may be modulated byphosphorylation through an alteration of net charge[22]. The DHN1 and DHN4 proteins from grapevinewere found to contain a higher number of putative PKCsites than CK2 sites, whereas DHN2 and DHN3 bore ahigher number of putative CK2 sites than PKC sites(Table 1; Figure 2). This finding is in agreement with aprevious suggestion that YnSKn-type DHNs are mainlyphosphorylated by PKC, while SKn-type DHNs aremainly phosphorylated by CK2 [22].DHN proteins with similar physicochemical properties

often also exhibit similar expression patterns. For ex-ample, while genes encoding alkaline YnSKn-type DHNs,such as At5g66400, HvDhn1, HvDhn2, HvDhn3,HvDhn4, HvDhn7, HvDhn9 and HvDhn10, are generally

induced by both embryogenesis and various types ofstress [18,23], those encoding acidic SKn- and KnS-typeDHNs, such as At1g20440, At1g20450, At1g76180,At1g5410, HvDhn8 and HvDhn13, are expressed consti-tutively in vegetative tissues and are also up-regulated byvarious types of stress [18,23,35]. The expression pat-terns of grapevine DHN1 and DHN2 agree with thosepredicted by their classification, which suggests that thisholds true in the species analyzed here.Even though the grapevine DHN1 and DHN4 (YnSKn-

type), as well as DHN2 and DHN3 (SKn-type), proteinsare grouped into only two classes, all four members ofthe grapevine DHN family exhibited very distinct pat-terns of expression (Figure 4, Figure 5, Figure 6, and Fig-ure 7). We found grapevine DHN1 to be induced bydrought, cold, heat, E. necator, and to be expressed dur-ing late stages of embryogenesis, which corresponds wellwith previous reports [28,29]. Conversely, DHN2 wasfound to be constitutively expressed in vegetative tissuesand was up-regulated under cold and heat conditions, aswell as during late embryogenesis (Figure 4, Figure 5,Figure 6, and Figure 7). In contrast, very low levels ofDHN3 expression were only detected during seed devel-opment with no induction observed in vegetative tissuesfollowing any of the stress or signaling molecule treat-ments studied here. Correspondingly, although noDHN3 transcripts could be identified in GenBank’s ESTdatabase, a large number (tens to hundreds) of theremaining grapevine DHN genes were (data not shown),which suggests that DHN3 is expressed at undetectablelevels in most tissue types. Likewise, DHN4 was also spe-cifically expressed during late embryogenesis, but at farhigher levels than DHN3 (Figure 4 and Figure 5). Theseresults suggest that the function of the grapevine DHNgenes is likely divergent, but may also exhibit some levelof overlap.The accumulation of DHNs in plants is believed to

have been associated with the acquisition of desiccation

Figure 8 Location of putative regulatory elements in the promoter regions of the V. yeshanensis and V. vinifera DHN genes. Promoterregions comprising 1500-bp of sequence upstream from the translational start sites were obtained from the published V. vinifera cv. Pinot Noirclone PN40024 genome sequence. In addition, the matching promoter regions were also cloned from V. yeshanensis acc. Yanshan-1 genomicDNA. Putative cis-regulatory elements were predicted using the PlantCARE website and those involved in stress-induction and seed developmentwere mapped. Recognition sequences are shown in Table 2.

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tolerance in these organisms [12] and expression levelsof these genes in vegetative tissues has generally beenfound to be higher in drought-tolerant cultivars than intheir susceptible counterparts [48-51]. However, this isnot always the case, as differences in expression levelsbetween tolerant and sensitive genotypes are oftendependent on the type of DHN, as well as the durationof the stress. While both V. yeshanensis and V. viniferahave been found to exhibit some tolerance to drought,the former exhibits a higher tolerance than the latterand also displays resistance to cold [25,26]. In the caseof induction via temperature stress, both DHN1 andDHN2 exhibited cold and heat responsiveness; however,DHN1 appeared to be far more responsive than DHN2(Figure 6 C-F). Interestingly, induction tended to be

higher in V. vinifera than V. yeshanensis, which is con-trary to the levels of temperature sensitivity in these twospecies.Conversely, among the four grapevine DHN genes

tested, only DHN1 was induced by drought stress invegetative tissues. This gene was up-regulated between1–2 d after the initiation of drought conditions in V. yes-hanensis, while its expression level at this time remainedunchanged in V. vinifera, suggesting that the expressionof DHN1 was quicker to respond to drought in the toler-ant genotype. However, V. yeshanensis did not show ahigher level of DHN1 expression than V. vinifera at 3and 4 d following treatment (Figure 6 A and B). A simi-lar situation has been observed in barley, where theHvDhn6 gene was expressed earlier in tolerant cultivars

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than susceptible cultivars under drought conditions, butat lower levels than the susceptible cultivars at timepoints that were further from the commencement ofdrought conditions [49,50].Generally, DHN genes are up-regulated under drought

stress and down-regulated following rehydration [52-55].However, in this study, the grapevine DHN1 and DHN2genes also displayed induction 2 h post-rehydration (Fig-ure 6 A and B). In line with this, it has been found previ-ously that leaf ABA content increases during earlyphases following re-hydration [56]. Therefore, the up-regulation of grapevine DHNs after rehydration may cor-respond to a change in leaf ABA content, since bothgenes were found to be responsive to this plant hormone(Figure 7 A and B).Recent studies have indicated that DHNs are also re-

sponsive to pathogen infection. For example, a DHNgene can be utilized to predict blast resistance in rice[57], and the Arabidopsis LTI30 and RAB18 genes havebeen found to be up-regulated by inoculation with pow-dery mildew [18]. This pathogen-induced expression ofDHNs may provide another important function for thistype of gene in disease resistance. In the current study,only DHN1 was found to be up-regulated in V. yesha-nensis and V. vinifera following inoculation with E. neca-tor, which is the causative agent of grapevine powderymildew (Figure 6G). Intriguingly, the expression level ofDHN1 was higher in the resistant V. yeshanensis than inthe susceptible V. vinifera, and a second induction eventwas also apparent in V. yeshanensis that was lacking inV. vinifera. These results suggest that DHN1 may par-ticipate in powdery mildew resistance in V. yeshanensis.DHN1 from V. vinifera was also induced by the signal-

ing molecules SA and MeJA, which are known to beinvolved in defense response, providing further evidencethat it could play a role in systemic acquired resistance[58]. It has been demonstrated previously that a numberof pathogen-responsive genes were up-regulated intransgenic Arabidopsis plants overexpressing DHN-5,which implies that DHNs might act as stress signalingmolecules that regulate defense genes [11]. This mayalso be the case for the DHN1 genes from grapevine(Figure 7A), although further experiments will be neces-sary to show this definitively.The expression of stress-responsive genes depends

upon the presence of cis-regulatory elements in theirpromoter regions [59], as has been shown to be the casefor barley DHN genes [32]. The four grapevine DHNpromoters exhibited distinct differences in the compos-ition and distribution of putative regulatory elementsheld within them. ABREs, which are one of the mostcommon cis-elements in the DHN promoters, likelyplayed a role in the induction of DHN1 by ABA, mediat-ing its expression under drought conditions. Indeed,

when taken together, all of the putative regulatory ele-ments identified within both the DHN1 and DHN2 pro-moters could account for their up-regulation by a varietyof stresses and their corresponding signal molecules(Figure 8). In contrast, relatively few regulatory elementswere found in the DHN3 and DHN4 promoters, whichcorresponds with the fact that neither of these geneswere found to be induced by any of the stresses or sig-naling molecules analyzed.The quantity and location of regulatory elements could

also have a profound effect on grapevine DHN expres-sion. It has been found previously that a single copy ofan ABRE is not sufficient for ABA-responsive inductionof transcription [59]. In this study, a higher number ofABRE elements were located in DHN1 and DHN2 pro-moters than in DHN3 and DHN4 promoters (Figure 8);correspondingly, the two former genes were responsiveto induction by ABA, whereas DHN3 and DHN4 werenot. Furthermore, the Skn-1 motif, which has beenshown previously to confer a promoter with endosperm-specific expression [60], was found in all four grapevineDHN promoters. However, these motifs were locatedmuch nearer to the translational start codon in DHN1and DHN4 promoters than in DHN2 and DHN3 promo-ters, which may provide an explanation for increasedup-regulation of DHN1 and DHN4 during late embryo-genesis (Figure 4 and Figure 5).

ConclusionsThe DHN gene family was identified in a genome-widesearch of the published genome sequence of V. vinifera,and the corresponding homologues were isolated fromV. yeshanensis. A large expansion of the DHN family ap-parently did not take place in grapevine, although it hasbeen a common occurrence in other plants. The fourgrapevine DHN genes shared a low sequence identity,and exhibited clear differences in physicochemical prop-erties and expression profiles, which indicates functionaldiversification within the grapevine DHN family. DHN1appeared to be the principal stress-responsive gene ingrapevine species, and was induced not only by variousabiotic stresses but also by E. necator. The small sizeand distinct expression profiles of the grapevine DHNgene family makes it an excellent model to elucidatefunctional differentiation within this gene family, whichshould contribute to the further understanding of thesegenes in plants.

MethodsPlant materialsV. yeshanensis acc. Yanshan-1 and V. vinifera cv.Pinot Noir were obtained from the Grapevine Re-pository of Northwest A&F University, Yangling,Shaanxi, China. One-year old rooted seedlings of

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Yanshan-1 and Pinot Noir were maintained in a green-house, and were utilized for stress experiments. For ex-pression analysis in different plant tissues, root, stem,leaf, seed and fruit peel samples were harvested fromthree representative veraison-stage V. vinifera and V.yeshanensis plants, respectively, that had been grownin the field. For expression analysis during seed develop-ment, flower buds were harvested from three Pinot Noirplants grown in the field 6 days before flower open-ing (−6 daf ). Flowers were collected at flower opening(0 daf ) and young berries were harvested at 6 daf. Inaddition, seeds were collected from 12–66 daf. Sampleswere frozen in liquid nitrogen and stored at −80°C.

RNA extraction and first-strand cDNA synthesisTotal RNA was isolated from plant tissues using themethod described by Reid et al. [61]. Subsequently, RNAwas treated with 10 units RQ1 RNase-free DNase (Pro-mega, Shanghai, China) in the presence of 100 unitsRNase inhibitor at 37°C for 30 min, followed by extrac-tion with phenol:chloroform:isoamyl alcohol (25:24:1)and chloroform:isoamyl alcohol (24:1). RNA was thenprecipitated with ethanol and dissolved in RNase-freewater. First-strand cDNA synthesis was carried out using1 μg total RNA and the PrimeScript™ RT reagent Kit(TaKaRa Biotechnology, Dalian, China) with 2.5 μMoligo dT primer and 2.5 μM random 6mer primer. Thereaction mixture was incubated at 37°C for 40 minand the reverse transcriptase was then inactivated at85°C for 5 s.

Cloning of DHN genes from V. yeshanensis and V. viniferaA fragment of the VyDHN1 coding sequence was ampli-fied from drought-treated leaves of V. yeshanensis acc.Yanshan-1 using differential display reverse-transcriptionPCR (DDRT-PCR) as described by Lin et al. [62]. Briefly,first-strand cDNA was synthesized from 1 μg total RNAisolated from leaves subjected to drought at 0, 2, 3, 4,and 5 d following onset of treatment using primerT11VA (V=A, C, G) at 37°C for 1 h with 200 units ofM-MLV (Promega) according to the manufacturer’sinstructions. This was followed by PCR amplificationwith primers T11VA and S476 (CCAAGCTGCC), fol-lowed by separation on a 6% polyacrylamide gel. Differ-ential fragments were recovered, amplified by a secondround of PCR using the same parameters as the first,and cloned into the pGEM-T easy vector (Promega).The 5’ end of the VyDHN1 cDNA was obtained by 5’rapid amplification of cDNA ends (5’ RACE) using theBD SMART RACE cDNA Amplification Kit (Clontech,CA, USA) with primer VD1-GSP1 (see Additional file 3for primer sequence) according to the manufacturer’srecommendations. The full-length VyDHN1 sequence

was then deduced through alignment of the originalDDRT-PCR fragment and 5’ RACE sequence.The encoded protein sequence of the VyDHN1 gene

was used to identify four V. vinifera DHN genes contain-ing putative K-segments via BLAST analyses. BLASTanalyses were also performed against the predictedgrapevine DHN genes using ten previously identifiedArabidopsis DHN proteins as query sequences [18].These results were further validated by searching forVitis DHN sequences in the Pfam database [63].For the remaining DHN genes, seeds were harvested

from V. yeshanensis at veraison. The DHN genes wereamplified from cDNA using PrimeSTARW HS DNApolymerase (TaKaRa) with primers that were designedbased upon the DHN genes of V. vinifera cv. Pinot Noir(see Additional file 3). Cycling parameters for amplifica-tion of VyDHN2 and VyDHN4 were as follows: 94°C for3 min, 25 cycles at 94°C for 30 s, 60°C for 30 s and 72°Cfor 30 s, followed by a final elongation at 72°C for 5 min.The same parameters were utilized in the case ofVyDHN3 except that 40 cycles were necessary for itsamplification. Subsequently, all four V. yeshanensis DHNgenes were also amplified from genomic DNA to identifyintronic regions using the same primers.V. yeshanensis DHN promoters, including 1500-bp of

upstream sequence in each case, were amplified fromgenomic DNA using PrimeSTARW HS DNA polymerase(TaKaRa) with primers that were designed based uponthe DHN genes of V. vinifera cv. Pinot Noir (see Add-itional file 4). Cycling parameters were as follows: 94°Cfor 3 min, 30 cycles at 94°C for 30 s, 60°C for 30 s and72°C for 2-3 min, followed by a final elongation at 72°Cfor 5 min. PCR products were cloned into pGEM-Teasy (Promoga) and three clones were sequenced perDHN gene.

Treatment of plants with various hormones, as well asabiotic and biotic stressDrought experiments were conducted by withholdingwater from V. yeshanensis and V. vinifera seedlings.Leaves were harvested 0, 0.5, 1, 2, 3, 4, 5, and 6 d follow-ing onset of treatment. Subsequently, the stressed plantswere watered to soil saturation and leaves were collected0.25, 0.5, 2, 6, 12, 24, and 48 h after watering. Plantsgrown under a normal watering regime were used as acontrol. For cold- and heat-stress induction,V. yeshanen-sis and V. vinifera seedlings were maintained in a growthchamber at either 4°C or 40°C and leaves were harvested0, 2, 6, 12, 24 and 48 h after treatment. Mock-treatedcontrol plants were kept in a growth chamber at 22°C. Pathogen treatment was carried out by inoculatingthe leaves of V. yeshanensis and V. vinifera with E.necator as previously described [27]. Prior to inocula-tion, leaves were sprayed with sterile water, and leaves

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were harvested 0, 0.5, 1, 2, 3, 4, 5, and 6 d after in-oculation. Control plants simply underwent the sterilewater spray and were not inoculated.For signaling molecule treatment, 100 μM ABA [29],

100 μM SA [64], and 50 μM MeJA [65] were sprayed onthe leaves of V. vinifera and leaf samples were harvested0, 1, 2, 4, 6, 8 h post-treatment. Leaves sprayed with0.05% Tween 20 solution were used as a negative con-trol. All stress-induction experiments were performedon three independent plants for each treatment.

Semi-quantitative and real-time RT-PCR analysisSemi-quantitative RT-PCR was performed using PremixEx TaqW Version2.0 (TaKaRa) and DHN-specific primersthat were designed to anneal to either side of an intronin both grapevine species (see Additional file 5 for pri-mer sequences). Genomic DNA was utilized as a sizecontrol for unspliced transcripts. Reactions lacking re-verse transcriptase were utilized as a negative control toexclude DNA contamination. Each experimental reac-tion (25 μl final volume) contained 1 μl of templatecDNA along with 400 nM of each primer. Cycling para-meters were as follows: 94°C for 3 min, 25 cycles at 94°Cfor 30 s, 60°C for 30 s and 72°C for 30 s, followed by afinal elongation at 72°C for 5 min. The actin1 transcript[GenBank:AF369524] was utilized as an internal controlusing primers designed to anneal to both V. vinifera andV. yeshanensis sequences and the same general para-meters as the DHN transcripts, except 20 cycles wereutilized for amplification. PCR products were separatedon a 1.5% agarose gel containing ethidium bromide, andphotographed using a Bio Imaging System (Syngene,Cambridge, UK). At least two technical replications wereconducted in each case.Real-time quantitative RT-PCR was conducted via the

ΔΔCT method using the SYBRW Premix Ex Taq II Kit(TaKaRa) with primer pairs designed to anneal withinthe second exon of each DHN gene in both grapevinespecies (see Additional file 6 for primer sequences). Thereactions were carried out in triplicate using 1 μl tem-plate cDNA in a final volume of 25 μl in an iQ5 RealTime PCR System (Bio-Rad, CA, USA) with the follow-ing thermal parameters: 95°C for 10 s, followed by40 cycles of 94°C for 5 s and 60°C for 30 s, with a finalmelting gradient from 60°C to 95°C at a rate of 1°C permin. The grapevine actin1 gene was utilized as an in-ternal control. Relative expression levels of each DHNgene from both species were analyzed using the IQ5software and were denoted as the fold-difference fromexpression present at baseline levels. Paired t-tests wereconducted using Origin Pro 8.0 software (OriginLabCorp., Northampton, MA, USA) to assess the signifi-cance of expression level differences between treatedsamples and the mock controls. Differences were

deemed significant at p < 0.05. Baseline expression signi-fies that which was present prior to treatment (ie. thefirst time point where relative expression is set to 1), ex-cept in the case of DHN3, where no expression wasnoted until 18 daf and therefore this time point was uti-lized as the baseline.

In silico analysis of DHN genes and their encoded proteinsChromosomal locations of VvDHN genes were predictedusing the BLAT server through the Genoscope GenomeBrowser (http://www.genoscope.cns.fr/blat-server/cgi-bin/vitis/webBlat). To identify putative cis-acting elementswithin the V. vinifera DHN promoters, contigs containingthe respective genes were obtained using BLAST. Inter-genic regions between the DHN genes and their upstreamgenes were determined according to annotations providedin GenBank. In the case of V. yeshanensis DHN promo-ters, sequences were obtained directly by cloning. Thepresence of regulatory elements in 1500-bp of sequenceupstream of each translational start codon was determinedusing the PlantCARE database [36].Protein MW (molecular weight), pI (isoelectric point)

and GRAVY (grand average of hydropathy) were pre-dicted using the ProtParam program (Expasy tools)based on their amino acid compositions. Predictions ofintrinsic disorder within each DHN gene from both spe-cies were conducted using the PONDR-FIT tool [66].Protein secondary structures were predicted using thePSIPRED v3.0 program [67]. The sequence algorithmNetPhosK (Expasy), with its probability limit set to 60%,was utilized to predict phosphorylation sites in VvDHNand VyDHN proteins.Phylogenetic analysis was carried out by performing

multiple alignments of full-length DHN proteinsequences from V. vinifera, V. yeshanensis, H. vulgareand Arabidopsis using MEGA5 [68]. DHN sequencesfrom Arabidopsis and barley were obtained from previ-ous reports [18,32-34]. An unrooted dendrogram wasconstructed based on the alignment with PhyML usingthe maximum likelihood method [69].

Additional files

Additional file 1: Cloning of the DHN1 coding sequence fromdrought-treated leaves of V. yeshanensis

Additional file 2: Structure prediction of DHN proteins from V.yeshanensis and V. vinifera

Additional file 3: Sequence of primers used for cloning DHN genesin grapevine

Additional file 4: Sequence of primers used for cloning DHNpromoters from V. yeshanensis

Additional file 5: Sequence of primers used for semi-quantitativeRT-PCR in grapevine

Additional file 6: Sequence of primers used for real-time qRT-PCR ingrapevine

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AbbreviationsABA: Abscisic acid; ABRE: ABA-responsive element; CK2: Casein kinase 2;Daf: Days after flowering; DDRT-PCR: Differential display reverse-transcriptionPCR; DHN: Dehydrin; Dpi: Days post inoculation; DRE: Dehydration-responsiveelement; HSE: Heat shock-responsive element; LTR: Low temperature-responsive element; MeJA: Methyl jasmonate; MeJA-RE: Methyl jasmonate-responsive element; NLS: Nuclear localization signal; PKC: Protein kinase C;qRT-PCR: Quantitative reverse-transcription PCR; SA: Salicylic acid; SnRK: Snf1-related kinase.

Authors’ contributionsYY contributed to the design of the study, conducted the majority ofexperiments and drafted the manuscript; MH contributed to the powderymildew treatment experiment; ZZ contributed to the signaling moleculetreatment experiment; SL contributed to the seed development experiment;YX and CZ were involved in the design of the study and preparation of themanuscript; SDS participated in analysis of results and preparation of themanuscript; YW conceived, designed and directed the study and contributedto the preparation of the manuscript. All authors read and approved the finalmanuscript.

AcknowledgementsThis work was supported by the National Natural Science Foundation ofChina (No.30971972) Nonprofit Finance Fund, as well as earmarked funds forModern Agro-industry Technology Research System (No. CARS-30-yz-7) andResearch of Non-profit Service (Agriculture Section) (No. 200903044–4).

Author details1College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100,China. 2Key Laboratory of Biology and Genetic Improvement of HorticulturalCrops (Northwest Region), Ministry of Agriculture, Northwest A&F University,Yangling, Shaanxi 712100, China. 3State Key Laboratory of Crop StressBiology in Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100,China. 4Department of Agricultural, Food and Nutritional Science, 4–10Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta T6G2P5, Canada.

Received: 28 February 2012 Accepted: 2 August 2012Published: 10 August 2012

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doi:10.1186/1471-2229-12-140Cite this article as: Yang et al.: Identification of the dehydrin genefamily from grapevine species and analysis of their responsiveness tovarious forms of abiotic and biotic stress. BMC Plant Biology 2012 12:140.